Protein kinases catalyze the phosphorylation of serine (Ser), threonine (Thr), and tyrosine (Tyr) residues in peptides and proteins, and comprise the largest superfamily of enzymes in eukaryotes with an estimated 500 genes in the human genome. Because most intracellular processes are regulated by protein phosphorylation, dysregulation of protein kinases can lead to a variety of disease states. Protein kinases are therefore important targets for drug discovery and development. Protein kinase-targeted drug discovery requires the ability to rapidly screen compounds for their ability to specifically inhibit selected protein kinases. To facilitate the high-throughput screening of kinase activities we will develop microplate protein kinase assays using a proprietary fluorescence technology developed at the University of Utah. First, we will prepare two sets of fluorescently labeled synthetic peptides. One set will be used to assay protein Ser/Thr kinases, the other for protein Tyr kinases. Second, we will optimize each protein kinase assay system for use in standard 96-well and 384-well fluorescence microplate readers. Based on feedback from outside evaluators, we will optimize and develop commercial versions of these protein kinase assay kits to be sold to the research community and companies involved in drug discovery and development.